It is often desirable to subject various liquids or other solutions to quantitative investigation by means of electrophoretic techniques, or other liquid separation techniques such as liquid chromatography. Many of these methods depend upon the measurement of the refractive index of the solution, and deduction of the distribution of the concentration of the relevant substances from the relative changes in refractive index monitor. Moreover, the trend in this industry has been driven by a consensus that smaller diameter flow columns provide higher separation efficiencies and reduce mobile phase usage.
To optimize the accuracy and reliability of the concentration monitoring devices and techniques, it is similarly necessary to utilize correspondingly small volumes of test fluids as well. The use of ever smaller capillary columns in various liquid separation techniques, however, has placed increased emphasis upon a need for miniaturization of refractive index detector technology. Apparatuses and techniques for measuring changes in the refractive index in small detection cells has not kept pace with the downsized capillary column developments.
For example, devices incorporating the interferometric method for determining the refractive index, such as devised by Mach and Zehnder, Michelson, and Jamin, are relatively complex. These methods require various plates and mirrors for separating and directing light beams through test and reference media, and sensitive adjustments which severely complicate and limit their applicability, especially in conjunction with small volume cells. U.S. Pat. No. 2,568,589, which issued to H. Labhart, attempted to address the shortcomings of the interferometric devices by providing a monochromatic light source directed against a plane parallel plate at an oblique angle to create two beams. A second plane parallel plate was arranged perpendicularly to the parallel light beams, and a test cell was arranged between the two plates and in line with one of the beams. The two beams were then compared by an optical system to produce an image of the test cell.
Similarly, U.S. Pat. No. 2,809,551, which issued to S. Svensson, utilizes an extended light source, and a half-transparent metal foil arranged between two congruent plane surfaces splits the beam of light so as to pass separately through a sample cell and a reference cell before being directed to a photographic plate. In addition to being relatively complex, these systems require two separate light beams and multiple passes of the light through the test medium held within a captive cell. More importantly, they were not readily adaptable to capillary separation schemes as required in modern operations.
An optical interferometer for high speed plasma diagnostics is shown in U.S. Pat. No. 3,539,262, which issued to T. Pryor. This device allegedly measures rapid phase changes in plasma due to electron density variations. Particularly, a laser beam is to be divided into two beams by a mirror beam splitter, and one of the beams passes through plasma held in a container. The other beam acts as a reference, and both beams are combined before passing through a slot and impinged on a photodiode. Electronics associated with the photodiode discriminate between the plasma light, noise, and phase shifted laser light passing through the plasma.
Similar split beam technology has also been applied to sample cuvettes for use with small volume capillary columns, such as for liquid chromatography and spectroscopic procedures. Particularly, a group of scientists from the University of Wyoming, recognizing that smaller volume capillary tubing produces a corresponding decrease in the detector volume, incorporated a beam splitter to direct a portion of a laser beam through a test cuvette having an inside diameter of approximately 100 micrometers, while passing the other beam directly to a reference photodiode. The first beam, having passed through the sample cell, was received by a photodiode identical with the reference photodiode, for a refractive index comparison. The cuvette was located on a three-axis translational stage to enable alignment with the laser beam, while the receiving photodiode was located on a single translation stage for movement perpendicular to the plane formed by the laser beam and the sample cuvette. The signal photodiode was then placed at the sharp boundary between the main beam and the first adjoining dark fringe created, and a strip chart recorder was utilized to monitor changes in the sample volume refractive index.
These experiments clearly recognized that the major limitation in capillary liquid chromatographic performance resided in the detector technology, and the difficulty in miniaturizing detector instrumentation without compromising its performance. Some of these same scientists performed other experiments at the University of Alberta, Edmonton, commenting upon the continuing problem in providing detector instrumentation having satisfactory performance with small tube capillary chromatography and electrophoresis. In these other tests, two separate laser light sources were provided and separately passed through a capillary test cuvette Capillary tubes having diameters of 50, 100 and 500 micrometers were tested, and a photodiode for monitoring the complicated beam profile was provided. The beam profile consisted of a set of diffraction fringes, and the photodiode was first located in the most intense portion of the beam profile, then translated perpendicularly to the beam axis such that the photodiode intensity was approximately 0.37 times the maximum intensity. It was recognized that while higher sensitivity could be obtained if the photodiode was receiving the most intense portion of the beam profile, linearity was compromised at higher intensity levels. Translation of the photodiode optimized sensitivity and linearity. It was found, however, that the smaller test cuvettes produced poorer refractive index performance, presumably due to temperature variations between the samples.
Consequently, while a great deal of effort has been directed to providing improved refractive index detection devices and procedures, heretofore there has not been provided a relatively universal detection device design which is easily adapted to small capillary diameters, which can provide adequate and dependable accuracy, and which can measure refractive index variations dynamically in a capillary flow tube used in liquid separation techniques.